https://doi.org/10.5607/en20049 Exp Neurobiol. 2020 Oct;29(5):334-343. pISSN 1226-2560 • eISSN 2093-8144 Short Communication Rho Guanine Nucleotide Exchange Factor 4 (Arhgef4) Deficiency Enhances Spatial and Object Recognition Memory Ki-Seo Yoo1, Kina Lee1, Yong-Seok Lee2, Won-Jong Oh3 and Hyong Kyu Kim1* 1Department of Medicine and Microbiology, Graduate Program in Neuroscience, College of Medicine, Chungbuk National University, Cheongju 28644, 2Department of Physiology, Department of Biomedical Science, Seoul National University College of Medicine, Seoul 03080, 3Neurovascular Unit Research Group, Korea Brain Research Institute, Daegu 41062, Korea Guanine nucleotide exchange factors (GEFs) play multiple functional roles in neurons. In a previous study, we reported that Arhgef4 (Rho guanine nucleotide exchange factor 4) functioned as a negative regulator of the excitatory synaptic function by sequestering postsynaptic density protein 95 (PSD-95). However, the role of Arhgef4 in behavior has not been examined. We performed comprehensive behavioral tests in knockout (KO) mice to investigate of the effects of Arhgef4 deficiency. We found that the expressed PSD-95 particle size was significantly increased in hippocampal neuronal cultures from Arhgef4 KO mice, which is consistent with the previous in vitro findings. Arhgef4 KO mice exhibited general motor activity and anxiety-like behavior comparable to those of the wild type littermates. However, spatial memory and object recognition memory were signifi- cantly enhanced in the Arhgef4 KO mice. Taken together, these data confirm the role of Arhgef4 as a negative synaptic regulator at the behavioral level. Key words: Arhgef4, PSD-95, Spatial memory, Recognition INTRODUCTION tin, a GEF of inhibitory synapses selectively activating the small GTPase Cdc42, results in a reduced capability of spatial learning Rho guanine nucleotide exchange factors (GEFs) are involved in and enhances anxiety-like behavior in collybistin-deficient mice the activation of Rho family GTPases by accelerating the exchange [4]. of GDP to GTP. Moreover, due to their multiple domains, GEFs Arhgef4, also known as Asef1, Adenomatous polyposis coli act as functional and structural regulators within the postsynaptic (APC)-stimulated GEF 1, was identified as a functional linker regions of neurons in response to external stimuli [1, 2]. Thus, protein connecting the tumor suppressor APC and G-protein GEFs play a crucial role in various behaviors, such as anxiety, signaling, suggesting its role in colon cancer [5]. Although Arhgef4 learning, and memory in experimental animals and also in human is highly expressed in the brain [6, 7], information on its function pathological conditions [2]. For example, the genetic deletion of in the brain is limited. In our previous study, we suggested that Kalirin7, a GEF of excitatory synapses for Rac1 and RhoG, shows Arhgef4 functions as a negative regulator in excitatory synapses, normal object recognition but impaired passive avoidance fear reducing the level of postsynaptic density protein 95 (PSD-95, also memory in Kalirin7 knockout (KO) mice [3]. The lack of collybis- known as synapse-associated protein 90), a major postsynaptic scaffolding protein [8]. PSD-95-deficiency causes memory impair- ment [9, 10]. The downregulation of Arhgef4 increases PSD-95, Submitted October 9, 2020, Revised October 12, 2020, but not Homer1, another postsynaptic density scaffolding protein. Accepted October 12, 2020 Conversely, the overexpression of Arhgef4 decreases PSD-95. Cor- *To whom correspondence should be addressed. respondingly, downregulation of Arhgef4 by RNA interference TEL: 82-43-261-2867, FAX: 82-43-272-1603 increases the frequency and amplitude of the miniature excitatory e-mail: [email protected] Copyright © Experimental Neurobiology 2020. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and www.enjournal.org reproduction in any medium, provided the original work is properly cited. Arhgef4 Inhibits Memory synaptic current (mEPSC) and its overexpression decreases them GluA1, and GluN1) of brain lysate from each genotyped mouse. [11]. However, these cellular and molecular studies were not All protein were separated on 8% acrylamide gels and transferred confirmed at the systemic or behavioral levels in experimental to a PVDF membranes (Millipore, Burlington, MA, USA). The animals. Consequently, in this study, we examined the behavior antibodies used in western blotting included rabbit polyclonal of Arhgef4 KO mice. Results showed that the ablation of Arhgef4 anti-Arhgef4 antibodies (raised by Arhgef4 N-terminal regions of improved spatial and recognition memories, again suggesting that 105 amino acids including APC-binding regions and SH domain, Arhgef4 functions as a synaptic negative regulator in the post- Accession# EDL14459; AbClone, Seoul, Korea), polyclonal anti- synaptic regions of excitatory synapses. PSD-95 antibodies (Cell Signaling Technology, Danvers, MA, USA), monoclonal anti-Homer 1 antibody (Synaptic Systems, MATERIALS AND METHODS Göttingen, Germany), polyclonal anti-GluA1 antibody (Millipore), monoclonal anti-GluN1 antibody (Millipore), polyclonal anti-syn- Transgenic animals aptophysin antibody (Millipore), and monoclonal anti-α-tubulin Arhgef4 KO mice (Arhgef4tm1a(KOMP)Wtsi, MGI Cat# 5782024, antibodies (Clone B-5-1-2, Sigma-Aldrich, St. Louis, MO, USA). RRID:MGI:5782024) were purchased from the National Institutes The protein levels of lysates were quantified by bicinchoninic acid of Health (NIH)-sponsored Knockout Mouse Program (KOMP assay (PierceTM BCA assay, Thermo Fisher Scientific, Rockford, repository, University of California at Davis, CA, USA) [12]. Het- IL, USA). All RT-PCR and western blot analyses were performed erozygotes (Arhgef4 +/tm1a(KOMP)Wtsi; Arhgef4 +/-) were crossed with more than twice to confirm reproducibility of data. heterozygotes to produce wild-type (WT, Arhgef4 +/+), heterozygous mutants (Hetero, Arhgef4 +/-), and homozygous mutants (Homo, Quantitative real-time PCR Arhgef4 -/-). Genotyping was performed using the indicated primer The 0.5 μg of total RNAs from the brain of WT, Hetero, or Homo sets according to the KOMP information. Further information was synthetized to cDNAs, and subsequently used to PCR con- on mouse generation and targeting strategies are available at the taining SYBR Green ready mix (TOPrealTM One-step RT qPCR Mouse Genome Informatics1 (MGI, http://www.informatics.jax. Kit, Enzynomics, Daejeon, Korea) and primers (identical sets used org) [13]. for RT-PCR analysis) by real-time PCR system (CFX96 Touch Real-Time PCR Detection System, Bio-Rad, Laboratory, Hercules, RT-PCR and western blotting CA, USA). The relative change of Arhgef4 expression in Homo or Arhgef4 deficiency in Arhgef4 KO mice was examined by RT- Hetero mouse to that in WT mouse was analyzed by 2-ΔΔCt method PCR and western blotting. For RT-PCR, 5 μg of the total RNAs [14, 15]. Total RNAs from two animals per genotype were sub- purified from the brains of WT, Hetero, and Homo were subjected jected to qRT-PCR analysis and the reactions were repeated. to reverse transcription using the oligo dT primer for 3’UTR of Arhgef4 mRNA, or a gene specific primer (Arhgef4-RT: 5’-gggcct- Neuronal culture, immunostaining, and image analysis gatggtataggcc-3) and SuperScript III (Cat# 18080-051, Invitrogen, Hippocampi were isolated from the brain of postnatal day one Carlsbad, CA) and subsequently amplified by PCR (primers for (P1) animals and used for culture as previously described [16]. Arhgef4 mRNA 3’-untranslated regions, Arhgef4-S: 5’-tccctg- After twelve days, the cultures were infected with Sindbis virus en- gttccaggttagtg-3’, Arhgef4-A: 5’-gcagccaggtcacttttcat-3’; β-actin coding green fluorescent protein (GFP) [11] for 12 h, followed by mRNA, β-actin-S: 5’-gcgcaagtactctgtgtgga-3’, β-actin-A: 5’-agcgc- immunostaining with monoclonal anti-PSD-95 antibody (Clone caaaacaaaacaaaa-3’; Arhgef4 mRNA coding regions: Arhgef4- 6G6-1C9, Thermo Fisher Scientific) and Cy3-conjugated goat 1500-S: 5’-ggaagccagaacagaagcag-3’, Arhggef4-coding-A: 5’-ggtt- anti-mouse IgG (Jackson ImmunoResearch Labs, West Grove, PA, gtctgatggatg-3’). Arhgef4 expression was examined on western USA). Fluorescent images were acquired with confocal microsco- blots containing 40 μg each of brain lysates from WT mice, Homo py (Zeiss LSM 800 Airyscan, Carl Zeiss Microscopy GmbH, Jena, mice, and rats (Sprague Dawley, Samtaco Bio Korea, Osan, Korea), Germany), and the acquired images were analyzed with the ImageJ and 10 μg of human embryonic kidney (HEK) cells. Synaptic program (ver 1.46r, NIH, Bethesda, MA, USA). Image acquisition protein expression was detected on western blots containing 3 μg and analysis were performed in blinded experiments and image (for synaptophysin and α-tubulin) or 25 μg (for PSD-95, Homer-1, analysis was performed as previously described [16]. The data are presented as mean±standard error of the mean (SEM). The Stu- 1 dent’s unpaired t-test was applied to reveal statistical differences http://www.informatics.jax.org/mgihome/nomen/IKMC_schematics. shtml (J:148605, J:173534) between the two groups. Statistical analyses were performed using https://doi.org/10.5607/en20049 www.enjournal.org 335 Ki-Seo Yoo, et al. GraphPad Prism (ver 5.0, GraphPad Software, La Jolla, CA, USA). Object location memory